The FBG domain of TNX interacts physically with the SLC in vitro and in vivo. (A) Quantitative (ELISA) analysis of mature human TGF-β1 associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF fragment, subjected (+) or not subjected (−) to conditions activating latent TGF-β (heat and acid treatments). (B) Levels of mature human TGF-β1 and of its LAP(β1) propeptide associated with equimolar concentrations (111 nM) of purified recombinant FBG domain, full-length TNX, or TNXΔEΔF protein were determined by immunoblotting. The monoclonal anti-TNX antibody recognizes the 10th FNIII domain. (C) Levels of phospho-Smad2 (P-Smad2) in NMuMG cells cultured for 3 h on noncoated dishes (N-C) or dishes coated with the different recombinant TNX variants (111 pmol/cm²) or stimulated with 5 ng/ml of soluble TGF-β1. Ratio of phospho-Smad2 to total Smad2/3 levels is indicated below. (D) Coimmunoprecipitation of the mature TGF-β1 entity and of its LAP(β1) propeptide with the purified recombinant FBG domain. Immunoprecipitations were performed with anti-FBG (α-FBG), anti-–human TGF-β1 (α-TGF-β1), or anti–human LAP(β1) antibodies or with control IgG. (E) Quantitative detection (ELISA) of mature TGF-β1 associated with the FBG domain of bovine TNX immunoprecipitated from FBS with either anti-FBG domain (α-FBG) antibody or control IgG. Samples were subjected (+) or not subjected (−) to activating conditions before the ELISA. An FBS fraction corresponding to 2.5% of the total volume used for the immunoprecipitation was also subjected to ELISA. *, P < 0.05. Error bars are means ± SD. IP, immunoprecipitation.